1. Field of the Invention
The present invention is directed to chemical compounds and methods useful in the regulation of thrombin receptor mediated cell stimulation. More specifically, the invention is directed to prothrombin-derived peptides and methods which employ such peptides for promoting wound healing and inhibiting scar formation, tissue adhesions, blood coagulation, tumor angiogenesis, tumor metastasis and pulmonary edema.
2. Description of the Related Art
Human alpha-thrombin appears to have growth-promoting activity for a wide variety of cells from various tissues. For example, alpha-thrombin has been shown to initiate proliferation of fibroblastic cells in culture without addition of serum or other purified growth factors, to synergize with epidermal growth factor in certain hamster fibroblasts and human endothelial cells, and to initiate cell division or DNA synthesis in mammalian lens epithelial and spleen cells. Yet, the use of thrombin as a growth factor and its potential importance to wound healing has not been widely acclaimed. In part, this may be due to the complexity of thrombin's involvement with coagulation, platelet activation, and initiation of cell proliferation as well as to the complex regulation of thrombin and thrombin-like molecules by serum protease inhibitors and by cell-released protease nexins. This complexity and high degree of physiologic regulation, however, supports the potential importance of this initiation pathway in wound healing.
Thrombin may also play a role in metastasis and angiogenesis of tumors. Generally, for a tumor to grow larger than a few millimeters in diameter, vascular endothelium must proliferate and form vesicle walls to provide circulation and nutrients to the cells inside of the tumor mass. Thrombin likely potentiates this process by virtue of its ability to induce proliferation of endothelial cells. In addition, thrombin has been shown to disrupt the normal intercellular endothelial cell contacts important in preventing cells and plasma factors from escaping or entering the microvasculature. The present hypothesis that thrombin may increase metastasis by disrupting these contacts is supported by studies demonstrating a correlation between decreased levels of anti-thrombin III (which removes thrombin and other proteases from plasma) and increased tumor metastasis.
Various studies have led the present inventors to conclude that high-affinity cell surface thrombin receptors (See Carney and Cunningham, Cell 15:1341, 1978) may be involved in tumor metastasis and anglogenesis. For example, studies have indicated that thrombin receptors can serve as binding sites for tissue plasminogen activator, a molecule secreted from metastatic tumor cells. Moreover, other studies demonstrate the involvement of tissue plasminogen activator in metastasis and anglogenesis. Thus, many of the effects of plasminogen activator may be mediated through its interaction with the cell surface thrombin receptor. It is therefore proposed that stimulation of the thrombin receptor serves to promote tumor metastases, while inhibition of the receptor will decrease metastatic activity.
Thrombin has also been shown to cause changes in the structure and function of cells which make up the endothelial vasculature. These studies suggest that thrombin may play a central role in the development of pulmonary edema as well as edema of other tissues. For example, thrombin has been shown to increase permeability of endothelial cell monolayers to macromolecules, to increase arterial pressure and pulmonary vascular resistance, to induce smooth muscle contraction, and to increase transcapillary fluid filtration. All of these effects may be mediated by thrombin's interaction with cell surface thrombin receptors.
A number of recent studies have attempted to elucidate the mechanisms for thrombin-mediated cell stimulation. These studies have indicated to the present inventors that initiation of cell proliferation by thrombin requires two signals. The first signal appears to be dependent upon binding of the thrombin molecule to the high affinity cell surface thrombin receptor, while the second signal results from the enzymic activity of the thrombin molecule. Thus, unlike alpha-thrombin, neither DIP-alpha-thrombin (a proteolytically inactive thrombin derivative that retains receptor-binding activity) nor gamma-thrombin (an esterolytically active, but non-binding thrombin derivative) can initiate DNA synthesis or cell division. However, simultaneous addition of these two non-mitogenic thrombin derivatives initiates a level of DNA synthesis and cell division comparable to that initiated by alpha-thrombin.
These same thrombin derivatives have been used to distinguish intracellular events stimulated by high-affinity thrombin receptor occupancy from those resulting from proteolytic cleavage. Alpha-thrombin and gamma-thrombin both stimulate Na.sup.+ /K.sup.+ ATPase activity, phosphoinositol turnover, and Ca.sup.2+ metabolism, whereas DIP-alpha-thrombin does not. Thus, these events are attributable to thrombin's enzymic activity, not to receptor occupancy. Furthermore, these signals (the release of diacylglycerol and inositol triphosphate to cause Ca.sup.2+ mobilization) may in turn activate protein kinase C. Accordingly, it has been shown that phorbol myristate acetate (PMA), which activates protein kinase C, can substitute for enzymically active gamma-thrombin and initiate cell division in the presence of receptor saturating levels of DIP-alpha-thrombin or monoclonal antibody to the thrombin receptor. Thus, the requirements for enzymically active thrombin may indirectly relate to its activation of protein kinase C.
The precise signals generated by high-affinity interaction of thrombin with its receptor have been more difficult to define. However, it has recently been shown that DIP-alpha-thrombin stimulates a transient increase in intracellular cAMP. In contrast to ion fluxes and phosphoinositide turnover, cAMP levels are maximally stimulated by DIP-alpha-thrombin but are not stimulated by gamma-thrombin. Attempts to replace DIP-alpha-thrombin with cAMP analogs, however, have been unsuccessful. Therefore, it is possible that thrombin receptor occupancy produces a number of signals in addition to changes in cAMP levels.
One problem associated with the clinical application of thrombin directly to achieve such benefits is its susceptability to protease inhibitors by serum anti-thrombins. Such problems have heretofore prevented the use of thrombin in the clinic and has led the present inventors to identify smaller thrombin-active and thrombin antagonistic polypeptides which are not sensitive to the inhibitory effects of thrombin inhibitors.
The present invention provides for a number of smaller polypeptides which have been tailored to interact with the thrombin receptor to selectively stimulate or inhibit thrombin receptor occupancy related signals. It is believed that these polypeptides will prove to be useful in a wide variety of clinical settings where successful recovery may be influenced by thrombin receptor-mediated events.